Objective To screen a panel of suitable microsatellite loci to analyze the genetic structure of experimental cat populations. Methods Seventy-four candidate microsatellite loci on 18 pairs of autosomes in cats were chosen from GenBank and related literature. Then, the PCR conditions were optimized by exploring the optimal annealing temperature. Based on the results of agarose gel electrophoresis and STR scanning, a set of microsatellites with clear and stable bands, abundant alleles, and even distribution on the 18 pairs of autosomes in cats were selected. Results Fiftyfive polymorphic and stably amplified STR loci were screened from 74 alternative microsatellite loci. Then, one to two higher polymorphic STR loci were selected in each autosome (other than A1 and B1 chromosomes) of the cat. Finally, 31loci with multiple alleles and even distribution on the 18 pairs of autosomes in cats were screened from 74 candidate microsatellite loci. Conclusions A set of microsatellite loci suitable for genetic structure analysis of cat populations is successfully screened.